从绵羊的成人和前卵巢收集的体外成熟卵母细胞的振动
Summary
该议定书旨在为成年和幼羊卵母细胞的玻璃化提供标准方法。它包括从体外成熟介质的准备到暖化后文化的所有步骤。卵母细胞在 MII 阶段使用 Cryotop 进行振动，以确保最低基本体积。
Abstract
在牲畜中，体外胚胎生产系统可以开发和维持，这要归功于大量卵巢和卵母细胞，这些卵巢和卵母细胞很容易从屠宰场获得。成人卵巢总是携带几个心脊囊，而在青春期前的捐赠者中，卵母细胞的最大数量在4周大时可用，而卵巢承担蚂蚁卵泡的峰值数量。因此，4周大的羔羊被认为是很好的捐赠者，即使与成年卵母细胞相比，前卵母细胞的发育能力较低。
从成人和前教皇捐赠者那里获得成功冷冻保存玻璃化卵母细胞的可能性将促进基础研究和商业应用。从前幼年捐赠者那里收集的卵母细胞的振动也允许缩短生成间隔，从而增加育种计划的遗传收益。然而，低温保存后发育潜力的丧失使得哺乳动物卵母细胞可能是冷冻保存最困难的细胞类型之一。在现有的冷冻保存技术中，玻璃化被广泛应用于动物和人类卵母细胞。尽管这项技术最近取得了进步，但暴露于高浓度的低温保护剂以及令人心寒的损伤和渗透应激仍然会导致一些结构和分子变化，并降低哺乳动物卵母细胞的发育潜力。在这里，我们描述了从青少年和成人捐赠者那里收集的羊卵母细胞的体外化协议，并在冷冻保存之前在体外成熟。该协议包括从卵母细胞体外成熟到体外成熟、变暖和后加热潜伏期的所有程序。在MII阶段振动的卵母细胞确实可以在变暖后受精，但它们需要额外的时间在受精前恢复由于冷冻保存程序造成的损害，并增加其发育潜力。因此，暖化后的文化条件和时间是恢复卵母细胞发育潜力的关键步骤，特别是当卵母细胞从青少年捐赠者那里收集时。
Introduction
长期储存雌性卵母细胞可以提供广泛的应用，如通过基因选择计划改善家畜繁殖，通过原地野生动物物种保护计划促进保护生物多样性，以及通过将储存的卵母细胞纳入体外胚胎生产或核移植计划1、2、3，促进体外生物技术的研究和应用。幼细胞振动也会通过缩短育种计划4的生成间隔来增加遗传收益。卵母细胞的超快速冷却和变暖的振动目前被认为是家畜卵母细胞冷冻保存的标准方法5。在反胃动物中，在体外化之前，卵母细胞通常是在体外成熟，从从屠宰场衍生的卵巢2中获取卵泡后。成人，特别是前腹卵巢4，6，确实可以提供几乎无限数量的卵母细胞冷冻保存。
在牛群中，在卵母细胞振动和变暖之后，在过去十年中，几个实验室通常报告的爆破细胞产量为>10%。然而，在小反胃动物卵母细胞化仍然被认为是相对较新的青少年和成人卵母细胞，和羊卵母细胞振动的标准方法仍有待确定2，5。尽管最近取得了进步，但玻璃化和加热的卵母细胞确实呈现出一些功能和结构上的改变，限制了它们的发展潜力7、8、9。因此，很少有文章报告在玻璃化/加热羊卵母细胞2的爆炸性发育在10%或以上。已研究了几种方法，以减少上述变化：优化玻璃化和解冻解决方案的组成10，11：试验使用不同的低温设备8，12，13：并在体外成熟（IVM）4、14、15和/或在变暖6后的恢复时间应用特定治疗。
在这里，我们描述了从青少年和成人捐赠者那里收集的羊卵母细胞的体外化协议，并在冷冻保存之前在体外成熟。该协议包括从卵母细胞体外成熟到体外化、变暖和后暖化培养期的所有程序。
Protocol
根据欧洲联盟第86/609/EC号指令和欧洲共同体委员会2007/526/EC的建议，动物议定书和下文所述的执行程序符合萨萨里大学生效的道德准则。 1. 为卵母细胞操纵准备介质 通过补充杜尔贝科的磷酸盐缓冲盐水0.1克/升青霉素和0.1克/升链霉素（PBS），为收集的卵巢的运输准备媒介。 通过稀释 9.5 克组织培养介质 （TCM） 199 粉末，用 1 升米利 Q 水补充青霉素 （0.1%） 来为?…
Representative Results
与成人捐赠者相比，青少年捐献者对卵母细胞的低温率较低。观察到的第一个效果是与成年卵母细胞相比，升温后的存活率较低（图1A：χ 2测试P<0.001）。幼细胞在变暖后表现出较低的膜完整性（图1B）。在成熟介质中使用树脂是为了验证这种糖是否能减少幼细胞的低温伤害。数据表明，23个卵母细胞成熟24小时，补充三甲糖素?…
Discussion
家畜卵母细胞低温保存不仅能长期保护雌性遗传资源，而且能促进胚胎生物技术的发展。因此，制定卵母细胞振动的标准方法将有利于牲畜和研究部门。在本议定书中，提出了一套完整的成年绵羊卵母细胞振动方法，可以代表为少年卵母细胞开发高效的卵母细胞振动系统的坚实起点。
建议方法的主要优点之一是它包括从卵母细胞收集、体外成熟、体外化和变暖的所有步骤。?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者没有获得这项工作的具体资金。玛丽亚·格拉齐亚·卡帕伊教授和瓦莱里亚·帕修博士因视频画外音和在视频制作过程中建立实验室而获得感谢。
Materials
| 2′,7′-Dichlorofluorescin diacetate | Sigma-Aldrich | D-6883 | |
| Albumin bovine fraction V, protease free | Sigma-Aldrich | A3059 | |
| Bisbenzimide H 33342 trihydrochloride (Hoechst 33342) | Sigma-Aldrich | 14533 | |
| Calcium chloride (CaCl2 2H20) | Sigma-Aldrich | C8106 | |
| Citric acid | Sigma-Aldrich | C2404 | |
| Confocal laser scanning microscope | Leica Microsystems GmbH,Wetzlar | TCS SP5 DMI 6000CS | |
| Cryotop Kitazato | Medical Biological Technologies | ||
| Cysteamine | Sigma-Aldrich | M9768 | |
| D- (-) Fructose | Sigma-Aldrich | F0127 | |
| D(+)Trehalose dehydrate | Sigma-Aldrich | T0167 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2438 | |
| Dulbecco Phosphate Buffered Saline | Sigma-Aldrich | D8537 | |
| Egg yolk | Sigma-Aldrich | P3556 | |
| Ethylene glycol (EG) | Sigma-Aldrich | 324558 | |
| FSH | Sigma-Aldrich | F4021 | |
| Glutamic Acid | Sigma-Aldrich | G5638 | |
| Glutaraldehyde | Sigma-Aldrich | G5882 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Glycine | Sigma-Aldrich | G8790 | |
| Heparin | Sigma-Aldrich | H4149 | |
| HEPES | Sigma-Aldrich | H4034 | |
| Hypoutarine | Sigma-Aldrich | H1384 | |
| Inverted microscope | Diaphot, Nikon | ||
| L-Alanine | Sigma-Aldrich | A3534 | |
| L-Arginine | Sigma-Aldrich | A3784 | |
| L-Asparagine | Sigma-Aldrich | A4284 | |
| L-Aspartic Acid | Sigma-Aldrich | A4534 | |
| L-Cysteine | Sigma-Aldrich | C7352 | |
| L-Cystine | Sigma-Aldrich | C8786 | |
| L-Glutamine | Sigma-Aldrich | G3126 | |
| LH | Sigma-Aldrich | L6420 | |
| L-Histidine | Sigma-Aldrich | H9511 | |
| L-Isoleucine | Sigma-Aldrich | I7383 | |
| L-Leucine | Sigma-Aldrich | L1512 | |
| L-Lysine | Sigma-Aldrich | L1137 | |
| L-Methionine | Sigma-Aldrich | M2893 | |
| L-Ornithine | Sigma-Aldrich | O6503 | |
| L-Phenylalanine | Sigma-Aldrich | P5030 | |
| L-Proline | Sigma-Aldrich | P4655 | |
| L-Serine | Sigma-Aldrich | S5511 | |
| L-Tyrosine | Sigma-Aldrich | T1020 | |
| L-Valine | Sigma-Aldrich | V6504 | |
| Magnesium chloride heptahydrate (MgSO4.7H2O) | Sigma-Aldrich | M2393 | |
| Makler Counting Chamber | Sefi-Medical Instruments ltd.Biosigma S.r.l. | ||
| Medium 199 | Sigma-Aldrich | M5017 | |
| Mineral oil | Sigma-Aldrich | M8410 | |
| MitoTracker Red CM-H2XRos | ThermoFisher | M7512 | |
| New born calf serum heat inactivated (FCS) | Sigma-Aldrich | N4762 | |
| Penicillin G sodium salt | Sigma-Aldrich | P3032 | |
| Phenol Red | Sigma-Aldrich | P3532 | |
| Polyvinyl alcohol (87-90% hydrolyzed, average mol wt 30,000-70,000) | Sigma-Aldrich | P8136 | |
| Potassium Chloride (KCl) | Sigma-Aldrich | P5405 | |
| Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | P5655 | |
| Propidium iodide | Sigma-Aldrich | P4170 | |
| Sheep serum | Sigma-Aldrich | S2263 | |
| Sodium azide | Sigma-Aldrich | S2202 | |
| Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | |
| Sodium dl-lactate solution syrup | Sigma-Aldrich | L4263 | |
| Sodium pyruvate | Sigma-Aldrich | P2256 | |
| Sperm Class Analyzer | Microptic S.L. | S.C.A. v 3.2.0 | |
| Statistical software Minitab 18.1 | 2017 Minitab | ||
| Stereo microscope | Olimpus | SZ61 | |
| Streptomycin sulfate | Sigma-Aldrich | S9137 | |
| Taurine | Sigma-Aldrich | T7146 | |
| TRIS | Sigma-Aldrich | 15,456-3 |
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Citer Cet Article
Succu, S., Serra, E., Gadau, S., Varcasia, A., Berlinguer, F. Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep. J. Vis. Exp. (173), e62272, doi:10.3791/62272 (2021).